苦荞种子胰蛋白酶抑制剂的分离纯化及部分性质研究

采用凝胶层析及离子交换层析等方法，从苦荞种子中分离出一组胰蛋白酶抑制剂（ＴＢＴＩ－Ⅰ、Ⅱ）．对其性质研究表明：两个组分均对胰蛋白酶有较强的抑制作用，对胰凝乳蛋白酶抑制作用较弱，其中ＴＢＴＩ－Ⅱ的抑制作用大于ＴＢＴＩ－Ⅰ，两者对胃蛋白酶、木瓜蛋白酶及枯草杆菌蛋白酶均无抑制作用．用ＳＤＳ－聚丙烯酰胺凝胶电泳和ＳｅｐｈａｄｅｘＧ－１００凝胶层析分别对纯化产物进行分析得出ＴＢＴＩ－Ⅰ和ＴＢＴＩ－Ⅱ的近似分子量分别为１５．０ｋＤ和１８．０ｋＤ．ＴＢＴＩ－Ⅰ、Ⅱ都具有较高的热稳定性，在１００℃处理１０ｍｉｎ后可保留８６％左右的抑制活性．ＴＢＴＩ在酸性环境下较为稳定，在ｐＨ２．０条件下保温１ｈ，仍保留７５％的抑制活性．用Ｌｉｎｅｖｅａｅｒ－Ｂｕｒｋ作图法得知，该抑制剂属竞争性抑制类型，ＴＢＴＩ－Ⅱ的Ｋｉ值为３．５９×１０－７ｍｏｌ／Ｌ（以ＢＡＰＮＡ为底物），对胰蛋白酶的摩尔抑制比为１∶１．４．

Purification and Some Properties of the Trypsin Inhibitor from Tartary Buckwheat Seeds

The proteinase inhibitor (TBTI Ⅰ、Ⅱ)from tartary buckwheat was isolated by trichloroacetic acid precipitation,salting out and chromatography on Sephadex G 75 and DEAE cellulose(DE 32).A series of kinetic studies on enzyme catalyzed reaction showed that the inhibitors from tartary buckwheat seeds had a strong inhibition on trypsin,slight inhibition on chymotrypsin,no inhibition on pepsin,papain and subtilisin.the components from DE 32 column were found to be homogeneous proteins,and the approximate molecular weights were 15 0 kD(TBTI Ⅰ)and 18 0 kD(TBTI Ⅱ)by SDS polyacrylamid gel electrophoresis and Sephadex G 100 column.TBTIs were stable to heat at 80℃ for 10 min,and up to 100℃ for 10 min,TBTI Ⅱ still had about 88% residual activities.When TBTI s were studied for the sensibility to various pH values,the inhibitors retained most activity within pH 2 0 6 0.Only about 15% inhibitory activity was retained after exposing to pH 12 0. The inhibition of trypsin by the TBTⅠ Ⅱ was competitive,with a K i value of 3 59×10 -7 mol/L (BAPNA as substrate) and the mole ratio was about 1∶1 4.